This proposal is focused on the metabolism and transport of mammalian glycosylphosphatidylinositols (GPIs), free glycolipids (i.e. non- protein-linked) that may serve as membrane anchors of proteins. GPI synthesis is blocked at the first step by a genetic defect in the clinical disorder paroxysmal nocturnal hemoglobinuria. Free GPIs are synthesized in the ER where transfer to protein occurs, but they are also able to migrate to other intracellular compartments including the plasma membrane (PM). The PM and probably the Golgi apparatus contain membrane domains resistant to Triton X-100 that are enriched in GPIs. The first aim investigates the presence of these Triton Insoluble Membranes (TIM) in the endoplasmic reticulum (ER) and proposes to characterize the composition of ER-TIM using ER markers as targets for immunopurification. The consequences of this type of membrane organization for GPI metabolism will be studied in the second aim. Our working hypothesis is that free GPIs sequestered in TIM in the ER are much less metabolically active than free GPIs outside TIM. Turnover rates of free GPIs within and outside TIM will be compared in intact cells and in ER membranes. In addition, the efficiency of in vitro mannosylation of GlcN-(acyl)PI in TIM and in non-TIM fractions of ER membranes from cells that accumulate this GPI will be examined. The third aim investigates the mechanisms involved in transporting GPIs from the ER to the PM. The effect of known blockers of vesicular traffic on the movement of radiolabeled GPIs will be tested in intact and permeabilized cells and in a reconstituted in vitro system consisting of purified ER membranes containing labeled GPIs as donor vesicles and purified PM/Golgi fractions as acceptor membranes. We will determine how exogenous lyso-alkyl-GlcN-PI is taken up by cells and in which intracellular compartments this GPI precursor is metabolized further in the GPI anchor pathway. A head group exchange activity that take places during metabolism of exogenous lyso-alkyl-GlcN-PI will be also characterized. These studies will provide a better understanding of the role played by compart-mentalization on the regulation of the GPI pathway.